With a large number of human genes now identified through the Human Genome Project, there is great interest in finding out how these genes act in concert to regulate the whole organism (Watson, Collins, 1995; Adams, et al., 1991, 1992; Cohen, et al., 1995; Chan, et al., 1994; Crabtree, et al., 1994).
The current focus of this research is in monitoring the genes' activities, i.e., levels of expression, as a function of cell type, cell condition, disease state, or drug therapy response. The general requirements of this type are severalfold:
First, the method must be able to handle large numbers of genes at once. Ideally, all or nearly all expressed genes from a given cell type may be represented.
Secondly, the method should be responsive to relatively small, as well as to large, changes in the levels of expression. For example, in monitoring the response of genes in a cell exposed to a given drug, it may be important to detect slight changes in levels of expression of genes, i.e., a two-fold increase or decrease in expression level, in order to identify drug-responsive genes. As another example, in studying the response of genes in a given disease state, e.g., tumor state, it may be important, in understanding the relationship between gene expression and disease state, to classify genes according to low, moderate, and high shifts in gene expression. More generally, the greater resolution that can be achieved, in terms of relative levels of expression, the more information that can be obtained from the studies.
Finally, the method should be amenable to small amounts of polynucleotide sample material, to obviate the need for amplification of sample material, with the attendant possibility of differential amplification.
A variety of methods for studying changes in gene expression have been proposed heretofore. One approach is based on differential hybridization between nucleic acid fractions from control and test sources. After hybridization of cDNA's from the two sources, those species “equally expressed” can be removed as DNA hybrids, leaving overexpressed or underexpressed cDNA's in single-stranded form. The single-stranded species can then be further characterized, e.g., by electrophoretic fractionation.
This approach has been useful as a starting point for isolation or characterization of polynucleotides of interest in a polynucleotide mixture, but is not suited to following small changes in expression levels, or tracking changes in a relatively large number of genes.
Another approach for comparing the relative abundances of polynucleotides in mixtures involves hybridizing individual labeled probe mixtures with different replicate filters containing immobilized gene sequences, e.g., colonies of cloned DNA. Colonies on the replicate filters which hybridize differentially to the two probe mixtures then represent gene sequences which are present in greater or lesser abundance in the two probe mixtures.
Because this method relies on comparing the amounts of label at corresponding positions on two different substrates (or filters), the resolution of the method is limited by (i) variations in the amount of immobilized DNA at corresponding array positions on the two different filters, (ii) variations in the extent of hybridization that occurs at corresponding array position, e.g., due to differences in probe accessibility to the immobilized DNA, and (iii) variations in measured reporter levels at corresponding array positions. To improve the resolution appreciably, one would have to average out these variations by conducting many hybridization measurements in parallel.
It would thus be useful to provide, for measuring the relative abundances of polynucleotides in complex mixtures, an improved method that avoids or overcomes the limitations above. In particular, the method should be adaptable to measuring the relative abundances, copy numbers or expression levels in a very large number (e.g., 50,000 to 100,000) of genes, at a high resolution, e.g., two-fold change in relative abundance, and at high sensitivity for rare genes. At the same time, the method should be simple to carry out.